Investigating the Transcriptomic and Expression Presence-Absence Variation Exist in Japanese Eel (Anguilla japonica), a Primitive Teleost.

The pan-genome was defined as the complete gene set across strains, and it is built upon genes displaying presence-absence variations (PAVs); the pan-transcriptome is defined by recalling the pan-genome. Indeed, a PAV is reflected from the expression presence-absence variation (ePAV). In this study, treated with androgen, eels, which are a primitive fish from the basal lineage of Teleost, with different ovarian developments were chosen and submitted to RAN-sequencing. Transcriptomes were the assembly against eel genome scaffolds; a pair was the unit (the same eel before and after treatment) to analyze DEGs (differentially expressed genes); the core, unique, or accessory genes were identified, and the list of DEGs was analyzed to investigate ePAV. The results suggest that there was ePAV in Japanese eel, and the ePAV of eel was analyzed by pathway enrichment. These results signify the importance of genetic differential expression on the variations of phenotypes by androgen, and a transcriptomic approach appears to enable extracting multiple layers of genomic data.

Sperm ultrastructure of Ruditapes variegata and Tapes literatus (Mollusca, Bivalvia, Veneridae, Tapetinae) from Pescadores, Taiwan.

In the present study, we have investigated the ultrastructures of the mature gonadal spermatozoa of R. variegata and T. literatus and presented comparisons with the Manila clam, R. philippinarum, sperm ultrastructure examined. Spermatozoa of R. variegata consist of (in anterior to posterior sequence): an elongate conical, deeply invaginated, acrosomal vesicle (length 1.58 ± 0.06 µm; width 0.99 ± 0.07 µm; invagination occupied by a granular subacrosomal material); a barrel-shaped nucleus (length 1.82 ± 0.06 µm; width 1.50 ± 0.03 µm); a midpiece consisting of two orthogonally arranged centrioles, surrounded by four spherical mitochondria; nine satellite fibers connecting the distal centriole to the plasma membrane; and a flagellum originating from the distal centriole. Contents of the acrosomal vesicle of R. variegata are differentiated into a very electron-dense basal ring and a less electron-dense zone (with seven dense transverse layers structure) on the anterior region of the acrosome. Spermatozoa of T. literatus differ from those of R. variegata and are characterized by a rounded-conical invaginated, acrosomal vesicle (length 0.88 ± 0.08 µm; width 0.77 ± 0.06 µm), with a basal ring; and an anteriorly-tapered, barrel-shaped nucleus (length 1.57 ± 0.04 µm; width 1.60 ± 0.09 µm); a midpiece composed of four mitochondria. Centriolar and flagellar details are essential as for R. variegata. Sperm morphology separating R. variegate, R. philippinarum, and T. literatus in different clades. The anterior region of the acrosomal vesicle in R. variegata sperm had the transverse bands structure whereas the apex of the acrosomal vesicle of T. literatus sperm had no such structure. This difference advocated that acrosomal feature could be an important character for taxonomic distinction. Our data supported the previous studies that the ultrastructure of bivalve sperm is species-specific. This advocates that the phyletic relationships of Tapetinae, commonly based on shell morphology, should also add additional and newer approaches.

Androgenic Sensitivities and Ovarian Gene Expression Profiles Prior to Treatment in Japanese Eel (Anguilla japonica).

Androgens stimulate ovarian development in eels. Our previous report indicated a correlation between the initial (debut) ovarian status (determined by kernel density estimation (KDE), presented as a probability density of oocyte size) and the consequence of 17MT treatment (change in ovary). The initial ovarian status appeared to be an important factor influencing ovarian androgenic sensitivity. We postulated that the sensitivities of initial ovaries are correlated with their gene expression profiles. Japanese eels underwent operation to sample the initial ovarian tissues, and the samples were stored in liquid nitrogen. Using high-throughput next-generation sequencing (NGS) technology, ovarian transcriptomic data were mined and analyzed based on functional gene classification with cutoff-based differentially expressed genes (DEGs); the ovarian status was transformed into gene expression profiles globally or was represented by a set of gene list. Our results also implied that the initial ovary might be an important factor influencing the outcomes of 17MT treatments, and the genes related with neuronal activities or neurogenesis seemed to play an essential role in the positive effect.

Correlation between the ovarian status and the androgen sensibility in the cultured Japanese eel, Anguilla japonica.

Different responsive abilities of different types of eels (cultured, cultured feminized, and wild silver eels) during artificial maturation are recognized, and maturity status at the beginning of artificial maturation might be important. Maturity may represented by the distribution pattern of oocyte diameters. Androgens have been demonstrated to stimulate ovarian development in eels. To determine the initial status, operations were performed on eels to identify sex and to sample ovarian tissue. The recovered eels were then treated with 17α-methyltestosterone (17MT), and the responses of individual eels to 17MT were determined by the fold change in the mean oocyte diameter before and after treatment. Sampled ovarian tissues were fixed in Bouin's solution, oocytes were isolated, and the diameter of isolated oocytes was measured. The ovarian status, determined by kernel density estimation (KDE), was presented by the probability density of measured oocyte diameters; compared with histograms, a description method, KDE, provided more subtle information on the investigated ovary. Our data indicated a correlation between the initial ovarian status (density pattern) and the consequence of treatment (change of ovary); we also argued the semelparity of the Japanese eel. Our results supported the hypothesis that the initial ovarian status is an important factor affecting artificial maturation and that androgens could ameliorate the initial status of the eel ovary.

Doping of transition metal dichalcogenides in molecularly imprinted conductive polymers for the ultrasensitive determination of 17ß-estradiol in eel serum.

Molecularly imprinted polymers (MIPs) have been developed to replace antibodies for the recognition of target molecules (such as antigens), and have been integrated into electrochemical sensing approaches by polymerization onto an electrode. Electrochemical sensing is inexpensive and flexible, and has demonstrated utility in point-of-care devices. In this work, several 2D (conductive) materials were employed to improve the performance of MIP sensors. Screen-printed electrodes were coated by the electropolymerization of aniline and metanilic acid, commingled with target molecules and various 2D materials. Tungsten disulfide (WS2) with an average particle size of 2 µm was found to increase the sensitivity of detection of molecularly imprinted conductive polymer-coated electrodes to 17ß-estradiol. As estradiol concentrations are important to eel aquaculture, we screened eel serum samples to determine their 17ß-estradiol concentrations, which were found to be in the range 28.2 ± 3.6 to 73.0 ± 11.6 pg/mL after dilution. These results were in agreement with measurements using commercial immunoanalysis.

Testosterone improves the transition of primary oocytes in artificial maturation eels (Anguilla japonica) by altering ovarian PTEN expression.

In mammals, androgens appear to enhance the development of primary ovarian follicles, but PI3K (phosphoinositide 3-kinases) pathway is well recognized as one of the critical pathways in early follicular development. Roles of the PI3K were revealed by deletion of PTEN (phosphatase and tensin homolog on chromosome 10). PTEN is demonstrated to play an important role in the early stage of follicle development. In the Japanese eel, two forms of PTEN have been cloned, but what their functions on the development of early ovarian follicles are still not clear. The natural blockage and inducible of ovarian development was a benefit to address this question in the eel. Testosterone (T) shows to ameliorate the early ovarian development in the eel. The aims of this study were to elucidate the two forms of PTEN by cellular and physiological criteria and to study the effects of T on the ovarian PTEN production in the exogenous pituitary extracts-stimulated eel. Our results suggested that two forms of PTEN are existing in the Japanese eel, and eel ovarian development corresponded to the decrease in ovarian PTEN expression, vice versa. In addition, the supplement of T on eel early ovarian development can be attributed to its PTEN inhibitor role.

Investigating expression profiles of VEGF-Flk, and Angpt1 during development of gas glands in Japanese eel (Anguilla japonica).

Angiogenesis is a highly regulated physiological process in animals. Angiopoietin-1 (Angpt1) induces the signaling pathways related to vessel maturation in late phase of angiogenesis, which recruits pericyte supplements to make compact interaction with vessel tubes. There are only few data showing Angpt1 functions in fish. By using degenerate primers, partial sequence (812 bp) of Angpt1 was cloned from Anguilla japonica, and deduced amino acids showed 80% similarity to those of zebrafish. Physiological functions of cloned eel Angpt1 were studied by in vitro and in vivo manipulations with gas glands (rete mirabile) taken as the tested target tissues. RT-PCR and immunofluorescent staining techniques were performed to examine the expression patterns of Angpt1 as well as VEGF-Flk. Experimental data showed that, in vitro, bFGF, PPAR beta agonist, and estradiol affected Angpt1 expression; while cobalt ions, a VEGF expression-inducer, did not affect Angpt1 expression. In vivo, expression levels of Angpt1 increased with body growth. Furthermore, Angpt1 expressions increased significantly in the late stage of gas glands in the stimulated eel. Successive expression patterns on VEGF-Flk, and Angpt1 on different development stages of gas glands were observed. Our results suggest that the original function of angiopoietin-1 on angiogenesis is conserved during evolution.

Cloning and characterization of ApRab4, a recycling Rab protein of Aiptasia pulchella, and its implication in the symbiosome biogenesis.

The biogenesis of Symbiodinium symbiosome in the host cells of the sea anemone, Aiptasia pulchella, involves retention of ApRab5 on and exclusion of ApRab11 from the organelle. One predicted consequence of this differential Rab association is the constant membrane fusion of symbiosomes with endocytic vesicles in the absence of parallel membrane retrieval and the subsequent formation of spacious symbiosomes, which nevertheless, contradicts the common perception. To solve this discrepancy, we determined whether membrane fusion occurs between symbiosomes and endocytic vesicles, and whether ApRab11-independent recycling is involved in symbiosome biogenesis. By using the biotin-avidin detection system, we found evidence for symbiosome-endocytic vesicle fusion. Cloning and characterization of ApRab4, an A. pulchella homolog of Rab4, showed that ApRab4 is associated with both the early endocytic and the perinuclear recycling compartments, and its normal function is required for the organization of the recycling compartments. Immunostaining localized ApRab4 to the symbiosome membrane, partially overlapping with ApRab5-decorated microdomains. Significantly, a treatment that impaired Symbiodinium photosynthesis also abolished symbiosome association of ApRab4. Furthermore, ApRab4 was quickly recruited to newly formed phagosomes, but prolonged association only occurred in those harboring live zooxanthelllae. We propose that ApRab4 retention on the symbiosome is an essential part of the mechanism for the biogenesis of Symbiodinium symbiosome.

ApRab3, a biosynthetic Rab protein, accumulates on the maturing phagosomes and symbiosomes in the tropical sea anemone, Aiptasia pulchella.

Symbiosome biogenesis and function are central to the endosymbiotic interaction between symbiotic dinoflagellates and their host cnidarians. To understand these important organelles, we have been conducting studies to identify and characterize symbiosome-associated proteins of the Rab family, key regulatory components of vesicular trafficking and membrane fusion in eukaryotic cells. Our prior studies have implicated three endocytic Rab proteins in the regulation of symbiosome biogenesis. Here, we show that ApRab3 is a new member of the Rab3 subfamily, associating with symbiosomes and accumulating on the maturing phagosomes in the A. pulchella digestive cells. ApRab3 is 78% identical to human Rab3C, and contains all Rab 3-specific signature motifs. EGFP-ApRab3-labeled vesicular structures tended to either align along the cell peripheral, or aggregate at one side of the nucleus. ApRab3 specifically co-distributed with the TGN marker, WGA, but not other organelle-specific markers tested. Immunofluorescence staining with a specific peptide antibody showed similar results. Significantly, an expression of a constitutively active mutant caused the enlargement and random dispersion of EGFP-ApRab3-decorated compartments in PC12 cells. Together, these data suggest that ApRab3 is a new member of the Rab3 subfamily, participating in the biosynthetic trafficking pathway, and symbiosome biogenesis involves an interaction with ApRab3-positive vesicles.

An endothelial-cell-enriched primary culture system to study vascular endothelial growth factor (VEGF A) expression in a teleost, the Japanese eel (Anguilla japonica).

A partial gene for eel (Anguilla japonica) vascular endothelial growth factor (VEGF) has been cloned and an endothelial-cell-enriched primary culture derived from rete mirabile established to study regulation of the expression of the eel VEGF gene. Cells were cultured in M199 medium containing 0.1% fetal calf serum (FCS) and serum-free M199 medium for long-and short-term experiments, respectively. Cells were separately treated with cobalt ions (Co2+), basic fibroblast growth factor (bFGF), and estradiol (E2), which have been demonstrated to stimulate mammalian VEGF A expression, followed by quantification of the VEGF mRNA levels by real-time reverse transcription polymerase chain reaction. Our results show that: (1) the deduced eel VEGF protein encoded by the cloned gene is about 130 amino acids in length, and is closely related to a zebrafish (Danio rerio) VEGF A; (2) the endothelial-cell-enriched rete mirabile primary culture containing mainly (over 70%) the capillary endothelial cells; (3) the expression levels of the eel VEGF transcript were increased by Co2+, bFGF, and E2 treatments in a dose-and time-dependent manner. Our data demonstrate that an eel partial VEGF gene has been cloned and its regulation of expression in endothelial-cell-enriched rete mirabile cell culture is similar to that in higher vertebrates.

ApRab11, a cnidarian homologue of the recycling regulatory protein Rab11, is involved in the establishment and maintenance of the Aiptasia-Symbiodinium endosymbiosis.

Endosymbiotic association of the Symbiodinium dinoflagellates (zooxanthellae) with their cnidarian host cells involves an alteration in the development of the alga-enclosing phagosomes. To uncover its molecular basis, we previously investigated and established that the intracellular persistence of the zooxanthella-containing phagosomes involves specific alga-mediated interference with the expression of ApRab5 and ApRab7, two key endocytic regulatory Rab proteins, which results in the selective retention of the former on and exclusion of the later from the organelles. Here we examined the role of ApRab11, a cnidarian homologue of the key endocytic recycling regulator, Rab11, in the Aiptasia-Symbiodinium endosymbiosis. ApRab11 protein shared 88% overall sequence identity with human Rab11A and contained all Rab-specific signature motifs. Co-localization and mutagenesis studies showed that EGFP-tagged ApRab11 was predominantly associated with recycling endosomes and functioned in the recycling of internalized transferrin. In phagocytosis of latex beads, ApRab11 was quickly recruited to and later gradually removed from the developing phagosomes. Significantly, although ApRab11 immunoreactivity was rapidly detected on the phagosomes containing either newly internalized, heat-killed zooxanthellae, or resident zooxanthellae briefly treated with the photosynthesis inhibitor DCMU, it was rarely observed in the majority of phagosomes containing either newly internalized live, or healthy resident, zooxanthellae. It was concluded that through active exclusion of ApRab11 from the phagosomes in which they reside, zooxanthellae interfere with the normal recycling process required for efficient phagosome maturation, and thereby, secure their intracellular persistence, and consequently their endosymbiotic relationship with their cnidarian hosts.

Photoperiod affects the development of central neurotransmitter systems of tilapia, Oreochromis mossambicus.

The effects of photoperiod on the development of central neurotransmitters were investigated with tilapia, Oreochromis mossambicus. Zero-day-old (the hatching day) tilapia were raised in three different photoperiods (light/dark cycle): 12/1, 24/0, and 0/24 h, respectively. On the 5th day, brain serotonin (5-HT), norepinephrine (NE), gamma-aminobutyric acid (GABA), and glutamate (Glu) contents were quantified by a high-performance liquid chromatograph with electrochemical detection. Similar experiments were performed on the 5-, 10-, 15-, 20-, and 25-day-olds. These results showed that the photoperiod influenced both brain NE and GABA contents during its respective restricted period, before days 10 posthatching. Brain 5-HT content was influenced, either facilitated or suppressed according to the developing stage, whereas, brain Glu content was not altered by the different photoperiod exposure throughout the present studies.

Serum estradiol-17beta and testosterone levels during silvering in wild Japanese eel Anguilla japonica.

To understand the changes of serum levels of sex steroids in the wild Japanese eel Anguilla japonica during silvering process, eels collected from the Kaoping River of Taiwan from August 2000 through June 2001 were examined. The maturational stages of female eels before and during silvering were divided into four stages: juvenile, sub-adult, pre-silver and silver stages based on skin coloration and oocyte diameter. Male eels were investigated only in the silver stage. Radioimmunoassays were employed to measure serum levels of estradiol-17beta (E(2)) and testosterone (T). The mean liver mass of the female eels increased significantly during silvering, but the mean hepatosomatic index remained constant. In contrast, mean ovarian mass and gonadosomatic index increased significantly during silvering. Serum concentrations of E(2) in females increased significantly during silvering (P<0.05), while E(2) was undetectable in silver males. The mean serum T concentrations increased significantly in females (P<0.05) during silvering, with lowest mean values in the juvenile stage and highest mean value in the silver stage. The mean serum T level in the silver males was significantly lower than in silver females (P<0.05). In conclusion, both serum E(2) and T concentrations increased with ovarian development of wild Japanese eels during silvering, while serum E(2) was undetectable in the silver male eels. The findings support the idea that androgen, but not estrogen, plays a major role in silvering process of the eels in both sexes.

Profiles of PGH-alpha, GTH I-beta, and GTH II-beta mRNA transcript levels at different ovarian stages in the wild female Japanese eel Anguilla japonica.

The complete complementary DNA (cDNA) encoding pituitary gonadotropin II-beta subunit (GTH II-beta) of Japanese eel Anguilla japonica was cloned and sequenced, and the profiles of pituitary glycoprotein hormone alpha subunit (PGH-alpha), GTH I-beta, and GTH II-beta mRNA transcript levels at different stages of ovarian development before vitellogenesis in the wild females were investigated. The maturity of female eels was divided into four stages: juvenile, sub-adult, pre-silver, and silver stages based on ovarian development and skin color. The GTH II-beta cDNA was cloned by reverse transcription and polymerase chain reaction (RT-PCR) amplification from total pituitary RNA. The full length GTH II-beta cDNA was obtained using 5(')- and 3(')-rapid amplification of cDNA ends. The cloned eel GTH II-beta cDNA consists of 646 bp nucleotides, including 53 bp nucleotides of 5(')-untranslated region (UTR), 423 bp of open reading frame, and 170 bp nucleotides of 3(')-UTR followed by a poly(A) tail. It encodes a 140-amino acid precursor molecule of GTH II-beta subunit with a putative signal peptide of 24 amino acids and a mature peptide of 116 amino acids. RT-PCR analysis showed that the pituitary transcript levels of alpha subunit steadily increased during eel silvering. The expression of GTH I-beta and II-beta mRNA levels, however, varied in different ovarian developmental stages. The mRNA expression of both GTH I-beta and GTH II-beta were detectable in juvenile stage. The expression levels of GTH II-beta mRNA, but not GTH I-beta, were significantly increased in sub-adult stage. The transcript levels of GTH I-beta and II-beta subunits further increased in pre-silver and silver stages. We demonstrated for the first time that the differential transcription patterns of pituitary PGH-alpha, GTH I-beta, and GTH II-beta mRNAs occur during silvering of the wild female Japanese eels.